Department for Immunochemistry and Glycobiology, Institute for the Application of Nuclear Energy-INEP, University of Belgrade, Banatska 31b, Zemun-Belgrade 11080, Serbia.
Asian J Androl. 2010 May;12(3):363-75. doi: 10.1038/aja.2009.98. Epub 2010 Feb 22.
Defining the molecular characteristics of seminal plasma proteins is essential for understanding their function in physiological and pathological conditions. Starting from the predicted importance of human seminal plasma gelatin-binding proteins, comprising fibronectin (FN) and FN-related molecules, for male fertility, this study aims at gaining insight into their immuno-glycobiochemical properties. Human seminal plasma from subjects with normal semen parameters were separated on a gelatin-Sepharose column and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using antibodies against distinct FN forms. Heterogeneity of the isolated molecular species was examined by protein chip arrays combined with surface-enhanced laser desorption/ionization time of flight mass spectrometry, on normal, metal and hydrophobic surfaces. Carbohydrate composition was investigated using mannose-, fucose- and sialic acid-specific plant lectins and galectin-1. The results obtained indicated a pattern of isolated proteins corresponding to that of known FN fragments, as confirmed by immunoreactivity. Among them heparin-binding ability was preferentially associated with low molecular mass species. As for posttranslational modifications, phosphorylation and glycosylation of distinct fragments were revealed. Lectin binding to fragments containing the gelatin-binding domain, particularly with Ricinus communis agglutinin I, was stronger than to fragments containing the cell-binding site of FN. A low level of sialylation and distinctive concanavalin A- and Lens culinaris agglutinin-reactive species were also observed. Galectin-1 did not interact with the isolated preparation. Resolving the molecular heterogeneity of normal human seminal plasma FN and gaining initial insight into possible similarities/differences with known FN molecular species may be considered a prerequisite step preceding challenging the clinical usefulness of these molecular properties.
定义精液蛋白的分子特征对于理解其在生理和病理条件下的功能至关重要。本研究从预测人类精液纤维结合蛋白（FN）和 FN 相关分子对男性生育力的重要性出发，旨在深入了解它们的免疫糖生化特性。从具有正常精液参数的受试者的精液中分离出的精液，在明胶-琼脂糖柱上分离，然后用针对不同 FN 形式的抗体进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和免疫印迹分析。使用蛋白质芯片阵列结合表面增强激光解吸/离子飞行时间质谱法，在正常、金属和疏水面上分析分离出的分子种类的异质性。使用甘露糖、岩藻糖和唾液酸特异性植物凝集素和半乳糖凝集素-1研究糖的组成。结果表明，分离出的蛋白质模式与已知的 FN 片段相对应，这通过免疫反应性得到了证实。其中肝素结合能力与低分子量物质优先相关。至于翻译后修饰，发现了不同片段的磷酸化和糖基化。凝集素与含有明胶结合域的片段结合的亲和力更强，特别是与蓖麻凝集素 I 的结合，而与 FN 的细胞结合位点的片段的结合亲和力较弱。还观察到低水平的唾液酸化和独特的伴刀豆球蛋白 A 和扁豆凝集素反应性物质。半乳糖凝集素-1与分离出的制剂没有相互作用。解析正常人精液 FN 的分子异质性并初步了解与已知 FN 分子种类的可能相似/差异，可以被视为挑战这些分子特性的临床实用性的前提步骤。
